Inhibitors of mammalian collagenase

ABSTRACT

Mammalian collagenase is inhibited by compounds having the formula ##STR1## and salts thereof, wherein R 1  is hydrogen or alkanoyl of 2 to 10 carbon atoms; 
     R 2  is hydroxy, amino, or ##STR2## R 3  is hydrogen, alkyl of 1 to 4 carbon atoms, ##STR3## R 4  is hydroxy, amino, arginine, leucine, glutamine, alanine or glycine; and 
     m is 0 or an integer of 1 to 9.

RELATED APPLICATION

U.S. patent application Ser. No. 25,701, filed Apr. 2, 1979 by Ondettiand Pluscec, discloses mercaptoacyldipeptides that are ACE inhibitors;i.e., in mammals, they inhibit the action of angiotensin convertingenzyme on angiotensin I, reducing the formation of angiotensin II.Angiotensin II has been implicated as a causative factor in variousforms of hypertension in mammals. The application discloses compoundshaving the formula ##STR4## wherein R_(a) can be, inter alia, hydrogenor alkanoyl; b is 0 or 1, R_(c) can be, inter alia, alkyl of 1 to 7carbon atoms; and A₁ and A₂ each is an α-amino or α-imino acid residuejoined through a peptide bond.

BACKGROUND OF THE INVENTION

The patent and non-patent literature both contain disclosures ofmercaptoacyl amido acid which are useful as ACE inhibitors (describedabove under "Related Application"). A representative disclosure is U.S.Pat. No. 4,053,651, which describes, inter alia, glycine derivativeshaving the formula ##STR5## wherein R_(d) can be, inter alia, hydrogenor alkanoyl; R_(e) can be, inter alia, alkyl of 1 to 7 carbon atoms; andR_(f) and R_(g) can be, inter alia, hydrogen.

U.S. Pat. No. 4,105,776 also discloses mercaptoacy amino acids. In thedisclosure of how to make the compounds of that invention intermediatesare disclosed having the formulas ##STR6## wherein, inter alia, R_(g)can be hydrogen or alkanoyl; R_(h) can be hydrogen; R_(i) can be alkylof 1 to 7 carbon atoms; R_(k) can be amino; and j can be 0, 1 or 2.

BRIEF DESCRIPTION OF THE INVENTION

Mammalian collagenase is inhibited by compounds having the formula##STR7## and salts thereof. In formula I, and throughout thespecification, the symbols are as defined below.

R₁ is hydrogen or alkanoyl of 2 to 10 carbon atoms;

R₂ is hydroxy, amino, or ##STR8##

R₃ is hydrogen, alkyl of 1 to 4 carbon atoms, ##STR9##

R₄ is hydroxy, amino, arginine, leucine, glutamine, alanine or glycine;and

m is 0 or an integer of 1 to 9.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formula I can be prepared using as a starting material2-(hydroxymethyl)-4-methylpentanoic acid. Heating the starting materialwith phosphonic acid yields 4-methyl-2-methylenepentanoic acid which canin turn be reacted with a thio acid having the formula

    R.sub.1 '-SH,                                              II

wherein R₁ ' is alkanoyl of 2 to 10 carbon atoms, to yield a producthaving the formula ##STR10## Treatment of a compound of formula III withconcentrated ammonium hydroxide yields the ammonium salt of the mercaptoacid having the formula ##STR11## which can be neutralized with acid.

The products of formula III can also be used to prepare the products offormula I wherein R₂ is amino. Conversion of an acid of formula III tothe corresponding amide having the formula ##STR12## can be accomplishedusing conventional techniques. For example, the acid can be convertedfirst to an acid halide, (e.g., by reaction with thionyl chloride) andsubsequently to an amide by ammonolysis. If concentrated ammoniumhydroxide is used for ammonlysis, the resulting product will be themercapto product having the formula ##STR13##

To prepare those compounds of formula I wherein R₂ is ##STR14## it isnecessary to first convert the 2-(hydroxymethyl)-4-methylpentanoic acidstarting material to the corresponding halogen substituted derivativehaving the formula ##STR15## wherein X is a halogen atom, preferablychlorine or bromine. The conversion can be accomplished using, forexample, a thionyl halide reagent.

Reaction of a compound of formula VII with an amino acid having theformula ##STR16## in the presence of alkali yields an intermediatehaving the formula ##STR17## After the completion of the above reaction,but before isolation of the product, it is desirable to add more alkalito the reaction mixture to form a compound having the formula ##STR18##A compound of formula X can be reacted with a thio acid of formula II toyield a product having the formula ##STR19## Treatment of a compound offormula XI with concentrated ammonium hydroxide yields an ammonium saltof the mercapto acid having the formula ##STR20## which can beneutralized with acid.

Those compounds of formula I wherein R₂ is ##STR21## can be preparedfrom the corresponding carboxylic acid of formula X. The carboxylic acidis first converted to an acyl halide, preferably an acid chloride, usingconventional techniques, e.g., reaction with a thionyl halide. Theresulting acyl halide, i.e., a compound having the formula ##STR22## canbe converted to the corresponding amide, i.e., a compound having theformula ##STR23## by ammonolysis. A compound of formula XIV can bereacted with a thio acid of formula II to yield a product having theformula ##STR24## The corresponding mercapto compound having the formula##STR25## can be prepared by treating an alkanoylthio compound offormula XV with concentrated ammonium hydroxide.

Those compounds of formula I wherein R₂ is ##STR26## and R₄ is arginine,leucine, glutamine, alanine or glycine can be prepared from thecorresponding carboxylic acid of formula XI. The carboxylic acid isfirst dissolved in an organic solvent, e.g., an aromatic hydrocarbonsuch as toluene, in the presence of an organic base. An alkylhalocarbonate such as ethyl chloroformate, is added to the solution andthe solution of the resulting mixed anhydride is then mixed with anaqueous solution of the appropriate amino acid or amino acid salt (i.e.,arginine leucine, glutamine, alanine or glycine) to yield the producthaving the formula ##STR27## wherein R₄ ' is arginine, leucine,glutamine, alanine or glycine. Treatment of a compound of formula XVIIwith concentrated ammonium hydroxide yields the corresponding mercaptocompound having the formula ##STR28##

The compounds of this invention form basic salts with various inorganicand organic bases which are also within the scope of this invention.Such salts include ammonium salts, alkali metal salts, alkaline earthmetal salts, salts with organic bases, e.g., dicyclohexylamine,benzathine, N-methyl-D-glucamine, hydrabamine and the like. Thepharmaceutically acceptable salts are preferred, although other saltsare also useful, e.g., in isolating or purifying the product.

The compounds of formula I have at least one asymmetric carbon atom; thecarbon noted with an asterisk (*) in formula I. The compoundsaccordingly exist in stereomeric forms or in racemic mixtures thereof.All of these are within the scope of this invention. The above describedsynthesis can utilize the starting compounds in the form of a racemicmixture or as a stereomer. When R₂ is an amino acid or a dipeptide, theL-isomers with respect to the carbon atom of the amino acids isgenerally preferred.

In mammals, collagenase is one of the key enzymes involved in thecartilage and joint destruction of rheumatoid arthritis; see, forexample, Arthritis and Rheumatism, 20 (6): 1231 (1977). It is,therefore, desirable to inhibit the action of the collagenase enzyme.

While not limiting the scope of this invention to a specific theory ormechanism of operation, it is nevertheless helpful to an understandingof the invention to review the possible reasons for the activity of thecompounds of formula I. The main components of cartilage are thecollagen polypeptide molecules. These polypeptides are cleaved bymammalian collagenase at a single site. The compounds of this inventionresemble the susceptible sequence of the collagen molecules and, it istheorized, bind to the mammalian collagenase enzyme and inhibit itsactivity.

The mammalian collagenase enzyme contains zinc, which assists in thecleavage of a glycine-leucine or glycine-isoleucine bond and contains anextended cleft which interacts with an extended portion of the collagenmolecule. This molecule in turn contains arginine as the last homologousamino acid in the substrate sequence adjacent to the cleavage site, asequence showing a high degree of homology among the various types ofcollagen molecules. The inhibitors of this invention make use of thesefeatures of the enzyme and make modifications to enhance binding to themammalian collagenase molecule.

The action of mammalian collagenase has also been implicated as acausative factor in several other diseases in mammals. These diseasesinclude periodontal disease, corneal ulceration, tumor invasiveness, andepidermolysis bullosa; see, for example, American Journal of Pathology,92 (2): 509 (1978) and The New England Journal of Medicine, 291 (13):652 (1974).

For use in the treatment of rheumatoid arthritis, the compounds of thisinvention can be administered to a mammal in need thereof either orallyor by injection intraarticularly into the affected joint. The dailydosage for a 70 kilogram mammal will be in the range of about 10milligrams to 1 gram.

The compounds of this invention can be formulated in compositions suchas tablets, capsules or elixirs for oral administration or in sterilesolutions or suspensions for parenteral administration. About 10 to 500mg of a compound of formula I or physiologically acceptable salt iscompounded with a physiologically acceptable vehicle, carrier,excipient, binder, preservative, stabilizer, flavor, etc., in a unitdosage form as called for by accepted pharmaceutical practice. Theamount of active substance in these compositions or preparations is suchthat a suitable dosage in the range indicated is obtained.

The following examples are specific embodiments of this invention.

EXAMPLE 1 2-(Mercaptomethyl)-4-methylpentanoic acid, ammonium salt (A)4-Methyl-2-methylenepentanoic acid

4-Methyl-2-(methylhydroxy)pentanoic acid (8.7 g, see Example 3 below) isheated with 10 drops of 85% phosphonic acid in a Wood's metal bath at220° C. for 20 minutes. A distillation head is attached and the pressureis slowly decreased to 60 mm while the temperature is increased to 270°C. Product starts to distill and the pressure is further decreased to 10mm. The vapor temperature varies between 180° and 190° C. The yield ofthe title compound as distillate is 7.0 g.

(B) 2-[(Acetylthio)methyl]-4-methylpentanoic acid

The above compound 6.8 g is stirred with 5 ml of thiolacetic acid underargon for 5 days. It is concentrated in vacuo and a portion isdistilled. The product boils at 117°-120° C. at 9 mm of Hg.

(C) 2-(Mercaptomethyl)-4-methylpentanoic acid, ammonium salt

The above product (0.8 g) is dissolved in 1.8 ml of concentratedammonium hydroxide at 5° C. under argon and stirred at 5° C. for 2hours. Product slowly crystallizes out. The reaction mixture islyopholized to 0.8 ml and the product is filtered to yield 0.4 g ofanalytical sample. Solid liquifies at 120°-130° C. with ammonia givenoff.

Analysis calc'd for C₇ H₁₇ O₂ SN: C, 46.90; H, 9.56; N, 7.81; S, 17.88.Found: C, 47.07; H, 9.73; N, 8.06; S, 17.45.

EXAMPLE 2 2-(Mercaptomethyl)-4-methylpentanamide (A)2-[(Acetylthio)methyl]-4-methylpentanoyl chloride

2-[(Acetylthio)methyl]-4-methylpentanoic acid (6.0 g) is combined withthionyl chloride (5 ml). The reaction mixture is allowed to exotherm to45° C. It is stirred at room temperature for about 16 hours and the acidchloride product is then vacuum distilled yielding 5.1 g, boiling point80°-90° C. at 10 mm of Hg.

(B) 2-(Mercaptomethyl)-4-methylpentanamide

2-[(Acetylthio)methyl]-4-methylpentanoyl chloride (0.8 g) is cooled to0° C. and the reaction flask is purged with argon. Concentrated ammoniumhydroxide (2.5 ml) is added and the reaction mixture is stirred at roomtemperature under argon for 5 hours. The oily material slowlysolidifies. Extraction with ethyl acetate yields 0.3 g of material whichcrystallizes upon standing for 16 hours under ethyl acetate yielding 0.2g of the title compound, melting point 147°-154° C.

Analysis calc'd for C₇ H₁₅ NOS: C, 52.14; H, 9.37; N, 8.69; S, 19.88.Found: C, 52.18; H, 9.05; N, 8.40; S, 19.44.

EXAMPLE 3 N-[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]glycine (A)Isocaproic Acid

Potassium cyanide (28 g) is partly dissolved in 125 ml of ethanol and 30ml of water. Amyl bromide (63.6 g) is added and the reaction mixture isdigested on the steam cone for 24 hours. The solution is decanted fromthe potassium bromide on to 35 g of potassium hydroxide. This isdigested on the steam cone for 20 hours, diluted with 50 ml of water andconcentrated in vacuo to remove the ethanol. A 1:1 mixture of sulfuricacid and water is added to the reaction mixture and product is extractedwith petroleum ether to yield 60.6 g of crude product. Vacuumdistillation yields 43.4 g of product boiling at 90°-98° C./9 mm of Hg.

(B) 2-(Hydroxymethyl)-4-methylvaleric acid

Diisopropylamine (20.6 g) is dissolved in 80 ml of dry tetrahydrofuran.This solution is cooled to -30° C. n-Butyllithium (77 ml of 2.6 M inhexane) is added dropwise in a nitrogen atmosphere at a rate thatmaintains the reaction at -30° to -20° C. This solution is stirred at-20° C. for 30 minutes. Isocaproic acid (11.6 g) in 10 ml oftetrahydrofuran is added dropwise at -20° to -10° C., then stirred at-10° C. for 30 minutes. In a separate flask, paraformaldehyde (28 g) isheated to about 200° C. and the vapors are carried in a stream ofnitrogen over the surface of the tetrahydrofuran solution of thedilithium salt of isocaproic acid. During this procedure the temperatureis kept between -10° and +10° C. After all of the paraformaldehyde hasvaporized the reaction mixture is cooled to 0° C. and 10% hydrochloricacid is added dropwise until the reaction mixture becomes acidic.Product is extracted with 2 portions of ether (400 ml each). The etheris dried with magnesium sulfate, filtered and concentrated in vacuo toyield 13.2 g of crude material. Product is vacuum distilled to yield 9.0g, boiling point 135°-142° C./9 mm of Hg.

(C) 2-(Chloromethyl)-4-methylvaleryl chloride

The above acid (9.0 g) is cooled to 0° C. Thionyl chloride (15 ml) isadded dropwise. After addition the reaction mixture is stirred for about16 hours. Product is distilled in vacuo to yield 6.8 g, boiling point42°-50° C./9 mm of Hg.

(D) N-[(2-Chloromethyl)-4-methyl-1-oxopentyl]glycine

Glycine (2.0 g) is dissolved in 10 ml of water containing 1.1 g (0.027mole) of sodium hydroxide. This solution is cooled to 5° C. and 15 ml of(1:3) toluene:tetrahydrofuran is added. The chloroacid chloride isdissolved in 20 ml of tetrahydrofuran and is added dropwise to theglycine solution at 5° C. The pH of the reaction mixture is moniteredand maintained at pH 8 to 9 by the addition of an aqueous solution of1.1 g of sodium hydroxide (volume 20 ml). After stirring for about 16hours the aqueous layer is separated, washed with ether and acidifiedwith 10% hydrochloric acid. Product is extracted with ethyl acetate,dried with magnesium sulfate, filtered and concentrated in vacuo toyield 6.0 g of product.

(E) N-(4-Methyl-2-methylene-1-oxopentyl)glycine

The above material is dissolved in 40 ml of 10% sodium hydroxide anddigested on the steam cone for 1 hour. The reaction mixture is cooled,neutralized with 10% hydrochloric acid and product is extracted withchloroform. The chloroform is dried with magnesium sulfate, filtered andconcentrated in vacuo. Product cyrstallizes to yield 4.3 g, meltingpoint 73°-80° C.

(F) N-[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]glycine

The above product is dissolved in 8 ml of thiolacetic acid and isstirred for about 16 hours under argon. The reaction mixture isconcentrated in vacuo eliminating excess thiolacetic acid. Product (3.9g) slowly crystallizes and is washed with hexane. Recrystallization fromisopropyl ether-ethyl acetate yields an analytical sample, melting point115°-122° C.

(G) N-[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]glycine

The above product (0.3 g) is dissolved in 0.5 ml of concentratedammonium hydroxide in a flask purged with argon. This solution isstirred at 5° C. for 20 minutes. The reaction mixture is treated with10% hydrochloric acid to pH 2, and product is extracted with ethylacetate. The ethyl acetate is dried with magnesium sulfate, filtered andconcentrated in vacuo. A sample is dried at 60° C. in vacuo overpotassium hydroxide and phosphorous pentoxide for 6 hours to yield thetitle compound.

Analysis calc'd for: C₉ H₁₇ NO₃ S C, 49.29; H, 7.81 N, 6.39; S, 14.62.Found: C, 49.44; H, 8.25; N, 6.51; S, 14.37.

EXAMPLE 4N-[6-[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]-1-oxohexyl]-L-arginine(A) 6-[(4-Methyl-2-methylene-1-oxopentyl)amino]hexanoic acid, sodiumsalt

2-(Chloromethyl)-4-methylvaleryl chloride (6.73 g; see Example 3C) isdissolved in 100 ml of toluene and is cooled to 0° C. 6-Aminohexanoicacid is dissolved in 35 ml of 1 N sodium hydroxide and is added to thevigorously stirred toluene solution. After 2 hours, 75 ml of 1 N sodiumhydroxide is added and the reaction mixture is stirred for about 16hours. The toluene is decanted and the aqueous solution is acidified andextracted with ether. The ether is dried with magnesium sulfate,filtered and concentrated in vacuo to yield 6.7 g of product. From NMRthis oil is shown to be a mixture of the chloro and dehydrohalogentatedproduct. This is dissolved in 15 ml of 10% sodium hydroxide and digestedon the steam cone for 1.5 hours. The aqueous solution is reacidified andextracted with ether. A sample (1.2 g) is removed and dissolved in 5 mlof 1 N sodium hydroxide. This solution is concentrated in vacuo and theresidue is washed with ether. The solid is washed with cold ethylacetate-ethanol and dried in vacuo to yield 1.0 g of product, meltingpoint 166°-171° C. The product was found to contain 1/4 mole of water.

(B) 6-[[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]amino]hexanoic acid

The free acid of the above salt (1.9 g) is dissolved in 100 ml ofbenzene. Thiolacetic acid (0.76 g) is added and the solution is stirredunder nitrogen for 48 hours. The reaction mixture is concentrated invacuo and water is added to the pot residue. Product is extracted withether and the ether is dried wth magnesium sulfate, filtered andconcentrated in vacuo to yield 1.9 g of product.

(C)N-[6-[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]-1-oxohexyl]-L-arginine

The above product (1.9 g) is dissolved in 150 ml of toluene and 0.66 gof triethylamine. This is cooled to 0° C. and 0.65 g of ethylchloroformate is added dropwise. After addition the reaction mixture isstirred at 0° C. for 45 minutes and then filtered through Celite into aflask containing 1.02 g of L-arginine in 25 ml of water. This is stirredfor 48 hours, and the aqueous layer is separated from the toluene andconcentrated in vacuo to yield 1.9 g of crude product which isredissolved in water and chromatographed with water through 200 g ofAvicel to remove unreacted arginine. After 500 ml of water has comethrough the column the product is eluted with 150 ml of water to yield1.65 g of material. This is chromatographed through 200 g of LH-20Sephadex. After 475 ml of water passed through the column, 10 mlfractions are collected. The chromatography is followed by spottingfractions on filter paper and developing colors for the two functionalgroups (guanidine and sulfhydryl). The first 6 fractions contain animpurity which gives a positive guanidine test but a negative S-acetyl.The S-acetyl is visualized by spraying with a 0.3% acetone solution of2,2'-dithiobis-(5-nitropyridine) then converting it to the sulfhydrylgroup by passing the filter paper over NH₃ vapors. The subsequentfractions totalling 150 ml yield 1.0 g of desired product uponconcentration in vacuo. (product softens 97°-120° C.). Analysisindicates the presence of 0.75 mole of water.

Analysis calc'd. for C₂₁ H₃₉ N₅ O₅ S.75 H₂ O: C, 51.78; H, 8.40; N,14.37; S, 6.58. Found: C, 51.70; H, 8.43; N, 14.60; S, 6.54.

EXAMPLE 5N-[6-[[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]amino]-1-oxohexyl]-L-arginine

One gram ofN-[6-[2-[(acetylthio)methyl]-4-methyl-1-oxopentyl]-1-oxohexyl-L-arginine(see Example 4) is dissolved in 3 ml of water. The solution is cooled to5° C. and argon is passed over the solution. Concentrated ammoniumhydroxide (1 ml) is added and the reaction mixture is stirred at 5° C.for 15 minutes. The solution is then lyophilized and the residue isstirred with acetonitrile (20 ml) containing 5 drops of water. The solidis filtered under argon and dried in vacuo over potassium hydroxide toyield 0.7 g of the title compound.

Analysis calc'd. for C₁₉ H₃₇ N₅ O₄ S.H₂ O: C, 50.75; H, 8.74; N, 15.57;S, 7.12. Found: C, 50.96; H, 8.84; N, 15.98; S, 6.76.

EXAMPLE 66-[[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]amino]hexanamide (A)6-[(4-Methyl-2-methylene-1-oxopentyl)amino] hexanamide

6-[(4-Methyl-2-methylene-1-oxopentyl)amino]hexanoic acid (2.8 g, seeExample 4A) is cooled to 5° C. and thionyl chloride is added (5 ml). Thereaction mixture is stirred at 35° C. for 3 hours, and concentrated invacuo at room temperature to remove excess thionyl chloride. The potresidue is cooled to 5° C. and concentrated ammonium hydroxide (50 ml)is added and stirred for 1 hour. Product is extracted with ethyl acetateand the ethyl acetate solution is dried over magnesium sulfate, filteredand concentrated in vacuo. The resulting viscous oil slowlycrystallizes, is washed with ether, and recrystallized from ethylacetate to yield 0.8 g of the title compound, melting point 76°-82° C.

(B) 6-[[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]amino]hexanamide

6-[(4-Methyl-2-methylene-1-oxopentyl)amino]hexanoic acid (0.7 g) isdissolved in 2 ml of thiolacetic acid and stirred under argon for 5days. The sample is concentrated in vacuo, dissolved in ethyl acetate,and washed with water. The ethyl acetate solution is dried withmagnesium sulfate in the presence of activated charcoal (to decolorize).Upon filtration and concentration in vacuo the product slowlycrystallizes and is washed with hexane. The product has a melting pointof 64°-80° C.

EXAMPLE 7N-[N-[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]glycyl]-L-arginine (A)N-[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]glycine, 4-nitrophenylester

N-[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]glycine (0.5 g) isdissolved in 30 ml of ethyl acetate along with p-nitrophenol (0.2 g).This solution is cooled to 5° C., N,N'-dicyclohexylcarbodiimide (0.4 g)is added portionwise and the reaction mixture is stirred at 5° for about16 hours. The dicyclohexyl urea is filtered off and the filtrate isconcentrated thoroughly in vacuo. Isopropyl ether is added to theresidue and allowed to sit for about 16 hours. The crystalline solid isfiltered to yield 0.4 g of product, melting point 95°-100° C.

(B) N-[N-[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]glycyl]-L-arginine

The above active ester (0.4 g) is dissolved in 50 ml of tetrahydrofuran(THF). This solution is cooled to 0° C., arginine (0.2 g) in 20 ml ofwater is added dropwise with vigorous stirring, and the reaction mixtureis stirred for about 16 hours. It is concentrated in vacuo to 20 ml andthe aqueous solution is washed with portions of ethyl acetate until thebright yellow color disappears from the aqueous phase. This isconcentrated to 4 ml and chromatographed on 160 g of cellulose with 1:4methanol:water. After 700 ml of solvent passes through the column,product elutes in 80 ml of solvent free of unreacted arginine. This islyophilized to yield 0.4 g of analytical product containing 0.5 mole ofwater.

Analysis calc'd for C₁₇ H₃₁ N₅ O₅ S.1/2H₂ O: C, 47.87; H, 7.56; N,16.42; S. 7.5. Found: C, 48.09; H, 7.73; N, 16.47; S. 7.6.

(C) N-[N-[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]glycyl]-L-arginine

The above product (0.4 g) is dissolved in 2 ml of water. The solution ispurged with argon and cooled to 5° C. Concentrated ammonium hydroxide (1ml) is added, the solution is stirred for 30 minutes and lyophilized forabout 16 hours. Acetonitrile (10 ml) is added to the residue followed by4 drops of water. This is stirred under argon until product becomes acolorless granular solid, which is filtered and dried in vacuo at 45° C.to yield 0.35 g of analytical sample, melting point 132°-146° C.

Analysis calc'd for C₁₅ H₂₉ N₅ O₄ S: C, 47.99; H, 7.79; N, 18.65; S,8.54. Found: C, 47.55; H, 7.99; N, 18.34; S, 8.30.

EXAMPLE 8N-[5-[[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]amino]-1-oxopentyl]-L-arginine(A) 5-[[2-(Chloromethyl)-4-methyl-1-oxopentyl]amino]pentanoic acid

5-Aminovaleric acid (3.2 g) is dissolved in 15 ml of water containing1.09 g of sodium hydroxide. This is cooled to 5° C. and2-(chloromethyl)-4-methylvaleryl chloride in 30 ml of THF is addeddropwise. The pH is maintained at 8-9 by the dropwise addition of 1.09 gsodium hydroxide in 20 ml of water as needed. After stirring for about16 hours the reaction mixture is diluted with 30 ml of water andconcentrated in vacuo to remove THF. The aqueous layer is washed withether, and acidified with 10% hydrochloric acid. Product is extractedwith ethyl acetate. The ethyl acetate is dried with magnesium sulfate,filtered and concentrated in vacuo to yield 4.3 g of product. Product ispurified by the solvent system 500 parts ether: 10 parts methanol: 5parts acetic acid, yielding 2.0 g of material.

(B) 5-[(2-Methylene-4-methyl-1-oxopentyl)amino]pentanoic acid

The above compound is dissolved in 10 ml of ethanol and 10 ml of 10%sodium hydroxide digested on the steam cone for 30 minutes, andconcentrated in vacuo to a volume of 10 ml. This solution if neutralizedwith 10% hydrochloric acid and product is extracted with ethyl acetate.The ethyl acetate is dried with magnesium sulfate, filtered andconcentrated in vacuo to yield 1.7 g of product.

(C) 5-[[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]amino]pentanoic acid

The above material is dissolved in 3 ml of thiolacetic acid and stirredat room temperature for about 16 hours. The reaction mixture is washedwith hexane and chromatographed on 100 g of silica using 50:5:2ether:methanol:acetic acid, yielding 0.8 g of material.

(D) 5-[[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]amino]pentanoicacid, 4-nitrophenyl ester

The above material is dissolved in 10 ml of ethyl acetate along withp-nitrophenol (0.37 g), the solution is cooled to 5° C. andN,N-dicyclohexylcarbodiimide (0.54 g) is added portionwise. The reactionmixture is stirred at 5° C. for about 16 hours. Dicyclohexyl urea isfiltered and the filtrate is concentrated in vacuo. The residue isdissolved in isopropyl ether and after standing for 2 hours it isfiltered free of additional dicyclohexyl urea and concentrated in vacuoto yield 1.0 g of active ester as an oil.

(E)N-[5-[[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]amino]-1-oxopentyl]-L-arginine

The above crude active ester is dissolved in 20 ml of THF, added to a 5ml aqueous solution of arginine (0.4 g) and stirred at room temperaturefor about 16 hours. The reaction mixture is concentrated in vacuo to 5ml and the aqueous solution is washed with ethyl acetate andchromatographed on 80 g of cellulose using 1:1 water:methanol. After 300ml of solvent elutes through the column, clean product elutes in a 20 mlfraction. Further portions contained contaminated product. Yield 0.25 gof analytical sample.

Analysis calc'd for C₂₀ H₃₇ N₅ O₅ S.1.7H₂ O: C, 48.82; H, 8.28; N,14.23; S, 6.54. Found: C, 48.72; H, 8.24; N, 14.67; S, 6.99.

(F)N-[5-[[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]amino]-1-oxopentyl]-L-arginine

The above sample is dissolved in 1 ml of water cooled to 5° C. andpurged with argon. Concentrated ammonium hydroxide (1 ml) is added, thereaction mixture is stirred at 5° C. for 30 minutes, lyophilized forabout 16 hours and washed with 10 ml of acetonitrile containing 4 dropsof water. Product is dried at 60° C. for 6 hours to yield an analyticalsample melting point 117°-130° C.

Analysis calc'd for C₁₈ H₃₅ N₅ O₄ S.H₂ O: C, 49.63; H, 8.56; N, 16.08;S, 7.36. Found: C, 49.56; H, 8.44; N, 15.94; S, 7.77.

EXAMPLE 9N-[8-[[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]amino]-1-oxooctyl]-L-arginine(A) 8-[[2-(Chloromethyl)-4-methyl-1-oxopentyl]amino]octanoic acid

8-Amino caprylic acid (4.3 g) is dissolved in 20 ml of water containing1.1 g of sodium hydroxide. To this is added 20 ml of THF, and 5 ml oftoluene, and the solution is cooled to 5° C.2-(Chloromethyl)-4-methylvaleryl chloride is dissolved in 10 ml of THFand added dropwise to the above solution. The pH of the reaction mixtureis maintained between 8 and 9 during the reaction time by the additionof a total of 1.1 g of sodium hydroxide in 20 ml of water, as needed.After stirring at room temperature for about 16 hours, the aqueous layeris separated and acidified with 10% hydrochloric acid. Product isextracted with ethyl acetate and the ethyl acetate is dried withmagnesium sulfate, filtered and concentrated in vacuo to yield 5.6 g ofproduct.

(B) 8-[(2-Methylene-4-methyl-1-oxopentyl)amino]octanoic acid

The above product is dissolved in 40 ml of absolute ethanol and 40 ml of10% sodium hydroxide, digested on the steam cone for 2 hours and cooledand acidified with 10% hydrochloric acid. Product is extracted withethyl acetate and the solution is dried with magnesium sulfate, filteredand concentrated in vacuo to yield 4.8 g of the product. The above tworeactions are repeated on the same scale to yield a total of 8.7 g ofunsaturated product.

(C) 8-[[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]amino]octanoic acid

The above product (8.2 g) is dissolved in 15 ml of thiolacetic acid,stirred under argon at room temperature for 48 hours, and concentratedin vacuo for 16 hours to remove excess thiolacetic acid. The residue isdissolved in ether and washed 4 times with water. The ether is driedwith magnesium sulfate, filtered and concentrated in vacuo to yield 8.9g. A small amount of impurity is washed away from the product withhexane. Yield 8.2 g.

(D) 8-[[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]amino]octanoic acid,4-nitrophenyl ester

The above compound (8.2 g) and p-nitrophenol (3.3 g) are dissolved in200 ml of ethyl acetate and cooled to 5° C.N,N'-Dicyclohexylcarbodiimide (5.0 g) is added portionwise and thereaction mixture is stirred at 5° C. for about 16 hours. Thedicyclohexylurea is filtered off and the ethyl acetate filtrate isconcentrated in vacuo for 1 hour. Diisopropyl ether (4 ml) and ethylacetate (4 ml) are added to the pot residue. A second crop ofdicyclohexylurea is obtained. The filtrate is concentrated in vacuo toyield 8.7 g of crude active ester.

(E)N-[8-[[2-[(Acetylthio)methyl]-4-methyl-1-oxopentyl]amino]-1-oxooctyl]-L-arginine

The active ester (4.4 g) is dissolved in 100 ml THF and added dropwiseto a precooled solution of arginine (1.7 g) in 20 ml of water. Thisreaction mixture is stirred for about 16 hours, diluted with 350 ml ofwater and concentrated in vacuo to about 370 ml. This aqueous solutionis washed with ethyl acetate until the bright yellow color disappears.During this procedure emulsion formation occurs which is broken up bythe addition of diatomaceous earth to the separatory funnel. The aqueouslayer is filtered and lyophilized. The crude residue (2.9 g) ischromatographed on 300 g of Avicel using 3:7 water:methanol. After 900ml of solvent passes through the column, product elutes in 100 ml ofsolvent to yield 1.0 g of analytical sample after drying 6 hours at 50°C. in vacuo, melting point 85°-102° C. Product contains 1/2 molarequivalent of water.

Analysis calc'd for C₂₃ H₁₃ N₅ O₅ S.1/2H₂ O: C, 54.10; H, 8.68; N,13.71; S, 6.28. Found: C, 54.36; H, 8.74; N, 13.46; S, 6.15.

(F)N-[8-[[2-(Mercaptomethyl)-4-methyl-1-oxopentyl]amino]-1-oxooctyl]-L-arginine

Due to limited water solubility the above product (0.65 g) is firstdissolved in 50 ml of argon purged ethanol followed by the addition of 2ml of concentrated ammonium hydroxide. This reaction mixture is stirredfor 1.5 hours at room temperature under argon. It is then lyophilized toalmost dryness. Acetonitrile is added and the reaction mixture isstirred until product becomes granular and white. It is filtered anddried in vacuo at 50° C. to yield analytical sample, melting point102°-130° C. Product contains 1/2 molar equivalent of water.

Analysis calc'd for C₂₁ H₄₁ N₅ O₄ S.1/2H₂ O: C, 53.82; H, 9.02; N,14.91; S, 6.84. Found: C, 54.17; H, 9.15, N, 15.02; S, 6.60.

What is claimed is:
 1. A method for reducing the adverse effects ofmammalian collagenase in a mammalian host in need thereof, whichcomprises administering to said mammal an effective amount of a compoundhaving the formula ##STR29## or a salt thereof wherein R₁ is hydrogen oralkanoyl of 2 to 10 carbon atoms;R₂ is hydroxy, amino or ##STR30## R₃ ishydrogen, alkyl of 1 to 4 carbon atoms, ##STR31## R₄ is hydroxy, amino,arginine, leucine, glutamine, alanine or glycine; and m is 0 or aninteger of 1 to
 9. 2. A method in accordance with claim 1 wherein themammalian host has rheumatoid arthritis.
 3. A method in accordance withclaim 1 wherein R₁ is hydrogen.
 4. A method in accordance with claim 1wherein R₁ is alkanoyl of 2 to 10 carbon atoms.
 5. A method inaccordance with claim 3 wherein R₂ is hydroxy.
 6. A method in accordancewith claim 3 wherein R₂ is amino.
 7. A method in accordance with claim 3wherein R₂ is ##STR32##
 8. A method in accordance with claim 7 wherein mis
 0. 9. A method in accordance with claim 7 wherein m is an integer of1 to
 9. 10. A method in accordance with claim 7 wherein R₃ is hydrogen.11. A method in accordance with claim 7 wherein R₃ is alkyl of 1 to 4carbon atoms.
 12. A method in accordance with claim 7 wherein R₃ is##STR33##
 13. A method in accordance with claim 7 wherein R₃ is##STR34##
 14. A method in accordance with claim 3 wherein R₄ is hydroxy.15. A method in accordance with claim 3 wherein R₄ is amino.
 16. Amethod in accordance with claim 3 wherein R₄ is arginine, leucine,glutamine, alanine or glycine.
 17. A method in accordance with claim 3wherein the compound administered is2-(mercaptomethyl)-4-methylpentanoic acid, ammonium salt.
 18. A methodin accordance with claim 3 wherein the compound administered is2-(mercaptomethyl)-4-methylpentanamide.
 19. A method in accordance withclaim 3 wherein the compound administered isN-[2-(mercaptomethyl)-4-methyl-1-oxopentyl]glycine.
 20. A method inaccordance with claim 3 wherein the compound administered isN-[6-[2-[(acetylthio)methyl]-4-methyl-1-oxopentyl]-1-oxohexyl]-L-arginine.21. A method in accordance with claim 3 wherein the compoundadministered isN-[6-[[2-(mercaptomethyl)]-4-methyl-1-oxopentyl]amino-1-oxohexyl]-L-arginine.22. A method in accordance with claim 3 wherein the compoundadministered is6-[[2-[(acetylthio)methyl]-4-methyl-1-oxopentyl]amino]hexanamide.
 23. Amethod in accordance with claim 3 wherein the compound administered isN-[N-[2-(mercaptomethyl)-4-methyl-1-oxopentyl]glycyl]-L-arginine.
 24. Amethod in accordance with claim 3 wherein the compound administered isN-[5-[[2-(mercaptomethyl)-4-methyl-1-oxopentyl]amino]-1-oxopentyl]-L-arginine.25. A method in accordance with claim 3 wherein the compoundadministered isN-[8-[[2-(mercaptomethyl)-4-methyl-1-oxopentyl]amino]-1-oxooctyl]-L-arginine.